Soft tissue and bone augmentation and bulking utilizing muscle-derived progenitor cells, compositions and treatments thereof

ABSTRACT

The present invention provides muscle-derived progenitor cells that show long-term survival following transplantation into body tissues and which can augment soft tissue following introduction (e.g. via injection, transplantation, or implantation) into a site of soft tissue. Also provided are methods of isolating muscle-derived progenitor cells, and methods of genetically modifying the cells for gene transfer therapy. The invention further provides methods of using compositions comprising muscle-derived progenitor cells for the augmentation and bulking of mammalian, including human, soft tissues in the treatment of various cosmetic or functional conditions, including malformation, injury, weakness, disease, or dysfunction. In particular, the present invention provides treatments and amelioration for dermatological conditions, gastroesophageal reflux, vesico-ureteral reflux, urinary incontinence, fecal incontinence, heart failure, and myocardial infarction.

This application is a divisional of U.S. application Ser. No. 11/505,734filed Aug. 17, 2006, which is a divisional of U.S. application Ser. No.09/549,937 filed Apr. 14, 2000 now U.S. Pat. No. 7,115,417, which is acontinuation-in-part of U.S. application Ser. No. 09/302,896 filed Apr.30, 1999 now U.S. Pat. No. 6,866,842, which claims priority to U.S.Provisional Application No. 60/083,917 filed May 1, 1998, which areincorporated herein by reference in their entireties.

GOVERNMENT INTEREST

This invention was made with government support under grant No. DE013420awarded by the National Institutes of Health. The government has certainrights in this invention.

FIELD OF THE INVENTION

The present invention relates to muscle-derived progenitor cells (MDC)and compositions of MDC and their use in the augmentation of bodytissues, particularly soft tissue and bone. In particular, the presentinvention relates to muscle-derived progenitor cells that show long-termsurvival following introduction into soft tissues and bone, methods ofisolating MDC, and methods of using MDC-containing compositions for theaugmentation of human or animal soft tissues and bone, includingepithelial, adipose, nerve, organ, muscle, ligament, and cartilagetissue. The invention also relates to novel uses of muscle-derivedprogenitor cells for the treatment of cosmetic or functional conditions,such as dermatological conditions, gastroesophageal reflux,vesico-ureteral reflux, urinary incontinence, fecal incontinence,skeletal muscle weakness, heart failure, and injury or weaknessassociated with myocardial infarction.

BACKGROUND OF THE INVENTION

Augmentation of soft tissue using synthetic materials such as siliconeor polytetrafluoroethylene (PTFE) is well known in the art. U.S. Pat.No. 5,876,447 to Arnett discloses the use of silicone implants forfacial plastic surgery. However, such synthetic materials are foreign tothe host tissue, and cause an immunological response resulting in theencapsulation of the implant and scarring of the surrounding tissues.Thus, the implant may produce additional functional or aestheticproblems.

Soft tissue augmentation using biopolymers such as collagen orhyaluronic acid has also been described. For example, U.S. Pat. No.4,424,208 to Wallace et al. discloses methods of augmenting soft tissueutilizing collagen implant material. In addition, U.S. Pat. No.4,965,353 to della Valle et al. discloses esters of hyaluronic acid thatcan be used in cosmetic surgery. However, these biopolymers are alsoforeign to the host tissue, and cause an immunological responseresulting in the reabsorption of the injected material. Biopolymers aretherefore unable to provide long-term tissue augmentation. Overall, theuse of biopolymers or synthetic materials has been wholly unsatisfactoryfor the purpose of augmenting soft tissue.

Soft tissue augmentation using cell-based compositions has also beendeveloped. U.S. Pat. No. 5,858,390 to Boss, Jr. discloses the use ofautologous dermal fibroblasts for the treatment of cosmetic andaesthetic skin defects. Although this treatment avoids the problemsinherent in the implantation or injection of synthetic materials orbiopolymers, it results in other complications. Because fibroblastsproduce collagen, the cells can cause the stiffening and distortion ofthe tissues surrounding the implant site.

The use of autologous fat cells as an injectable bulking agent has alsobeen described (For review, see K. Mak et al., 1994, Otolaryngol. Clin.North. Am. 27:211-22; American Society of Plastic and ReconstructiveSurgery: Report on autologous fat transplantation by the ad hoccommittee on new procedures, 1987, Chicago: American Society of Plasticand Reconstructive Surgery, A. Chaichir et al., 1989, Plast. Reconstr.Surg. 84: 921-935; R. A. Ersek, 1991, Plast. Reconstr. Surg. 87:219-228;H. W. Horl et al., 1991, Ann. Plast. Surg. 26:248-258; A. Nguyen et al.,1990, Plast. Reconstr. Surg. 85:378-389; J. Sartynski et al., 1990,Otolaryngol. Head Neck Surg. 102:314-321. However, the fat graftingprocedure provides only temporary augmentation, as injected fat isreabsorbed into the host. In addition, fat grafting can result in noduleformation and tissue asymmetry.

Myoblasts, the precursors of muscle fibers, are mononucleated musclecells that fuse to form post-mitotic multinucleated myotubes, which canprovide long-term expression and delivery of bioactive proteins (T. A.Partridge and K. E. Davies, 1995, Brit. Med. Bulletin 51:123-137; J.Dhawan et al., 1992, Science 254: 1509-12; A. D. Grinnell, 1994, MyologyEd 2, A. G. Engel and C. F. Armstrong, McGraw-Hill, Inc., 303-304; S.Jiao and J. A. Wolff, 1992, Brain Research 575:143-7; H. Vandenburgh,1996, Human Gene Therapy 7:2195-2200).

Cultured myoblasts contain a subpopulation of cells that show some ofthe self-renewal properties of stem cells (A. Baroffio et al., 1996,Differentiation 60:47-57). Such cells fail to fuse to form myotubes, anddo not divide unless cultured separately (A. Baroffio et al., supra).Studies of myoblast transplantation (see below) have shown that themajority of transplanted cells quickly die, while a minority survive andmediate new muscle formation (J. R. Beuchamp et al., 1999, J. Cell Biol.144:1113-1122). This minority of cells shows distinctive behavior,including slow growth in tissue culture and rapid growth followingtransplantation, suggesting that these cells may represent myoblast stemcells (J. R. Beuchamp et al., supra).

Myoblasts have been used as vehicles for gene therapy in the treatmentof various muscle- and non-muscle-related disorders. For example,transplantation of genetically modified or unmodified myoblasts has beenused for the treatment of Duchenne muscular dystrophy (E. Gussoni etal., 1992, Nature, 356:435-8; J. Huard et al., 1992, Muscle & Nerve,15:550-60; G. Karpati et al., 1993, Ann. Neurol., 34:8-17; J. P.Tremblay et al., 1993, Cell Transplantation, 2:99-112; P. A. Moisset etal., 1998, Biochem. Biophys. Res. Commun. 247:94-9; P. A. Moisset etal., 1998, Gene Ther. 5:1340-46). In addition, myoblasts have beengenetically engineered to produce proinsulin for the treatment of Type 1diabetes (L. Gros et al., 1999, Hum. Gen. Ther. 10:1207-17); Factor IXfor the treatment of hemophilia B (M. Roman et al., 1992, Somat. Cell.Mol. Genet. 18:247-58; S. N. Yao et al., 1994, Gen. Ther. 1:99-107; J.M. Wang et al., 1997, Blood 90:1075-82; G. Hortelano et al., 1999, Hum.Gene Ther. 10:1281-8); adenosine deaminase for the treatment ofadenosine deaminase deficiency syndrome (C. M. Lynch et al., 1992, Proc.Natl. Acad. Sci. USA, 89:1138-42); erythropoietin for the treatment ofchronic anemia (E. Regulier et al., 1998, Gene Ther. 5:1014-22; B. Dalleet al., 1999, Gene Ther. 6:157-61), and human growth hormone for thetreatment of growth retardation (K. Anwer et al., 1998, Hum. Gen. Ther.9:659-70).

Myoblasts have also been used to treat muscle tissue damage or disease,as disclosed in U.S. Pat. No. 5,130,141 to Law et al., U.S. Pat. No.5,538,722 to Blau et al., and application U.S. Ser. No. 09/302,896 filedApr. 30, 1999 by Chancellor et al. In addition, myoblast transplantationhas been employed for the repair of myocardial dysfunction (C. E. Murryet al., 1996, J. Clin. Invest. 98:2512-23; B. Z. Atkins et al., 1999,Ann. Thorac. Surg. 67:124-129; B. Z. Atkins et al., 1999, J. Heart LungTransplant. 18:1173-80).

In spite of the above, in most cases, primary myoblast-derivedtreatments have been associated with low survival rates of the cellsfollowing transplantation due to migration and/or phagocytosis. Tocircumvent this problem, U.S. Pat. No. 5,667,778 to Atala discloses theuse of myoblasts suspended in a liquid polymer, such as alginate. Thepolymer solution acts as a matrix to prevent the myoblasts frommigrating and/or undergoing phagocytosis after injection. However, thepolymer solution presents the same problems as the biopolymers discussedabove. Furthermore, the Atala patent is limited to uses of myoblasts inonly muscle tissue, but no other tissue.

Thus, there is a need for other, different soft tissue augmentationmaterials that are long-lasting, compatible with a wide range of hosttissues, and which cause minimal inflammation, scarring, and/orstiffening of the tissues surrounding the implant site. Accordingly, themuscle-derived progenitor cell-containing compositions of the presentinvention are provided as improved and novel materials for augmentingsoft tissues. Further provided are methods of producing muscle-derivedprogenitor cell compositions that show long-term survival followingtransplantation, and methods of utilizing MDC and compositionscontaining MDC to treat various aesthetic and/or functional defects,including, for example, dermatological conditions or injury, and muscleweakness, injury, disease, or dysfunction.

It is notable that prior attempts to use myoblasts for non-muscle softtissue augmentation were unsuccessful (U.S. Pat. No. 5,667,778 toAtala). Therefore, the findings disclosed herein are unexpected, as theyshow that the muscle-derived progenitor cells according to the presentinvention can be successfully transplanted into non-muscle and musclesoft tissue, including epithelial tissue, and exhibit long-termsurvival. As a result, MDC and compositions comprising MDC can be usedas a general augmentation material for muscle or non-muscle soft tissueaugmentation, as well as for bone production. Moreover, since themuscle-derived progenitor cells and compositions of the presentinvention can be derived from autologous sources, they carry a reducedrisk of immunological complications in the host, including thereabsorption of augmentation materials, and the inflammation and/orscarring of the tissues surrounding the implant site.

Although mesenchymal stem cells can be found in various connectivetissues of the body including muscle, bone, cartilage, etc. (H. E. Younget al., 1993, In Vitro Cell Dev. Biol. 29A:723-736; H. E. Young, et al.,1995, Dev. Dynam. 202:137-144), the term mesenchymal has been usedhistorically to refer to a class of stem cells purified from bonemarrow, and not from muscle. Thus, mesenchymal stem cells aredistinguished from the muscle-derived progenitor cells of the presentinvention. Moreover, mesenchymal cells do not express the CD34 cellmarker (M. F. Pittenger et al, 1999, Science 284:143-147), which isexpressed by the muscle-derived progenitor cells described herein.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide novel muscle-derivedprogenitor cells (MDC) and MDC compositions exhibiting long-termsurvival following transplantation. The MDC of this invention andcompositions containing the MDC comprise early progenitor muscle cells,i.e., muscle-derived stem cells, that express progenitor cell markers,such as desmin, M-cadherin, MyoD, myogenin, CD34, and Bcl-2. Inaddition, these early progenitor muscle cells express the Flk-1, Sca-1,MNF, and c-met cell markers, but do not express the CD45 or c-Kit cellmarkers.

It is another object of the present invention to provide methods forisolating and enriching muscle-derived progenitor cells from a startingmuscle cell population. These methods result in the enrichment of MDCthat have long-term survivability after transplantation or introductioninto a site of soft tissue. The MDC population according to the presentinvention is particularly enriched with cells that express progenitorcell markers, such as desmin, M-cadherin, MyoD, myogenin, CD34, andBcl-2. This MDC population also expresses the Flk-1, Sca-1, MNF, andc-met cell markers, but does not express the CD45 or c-Kit cell markers.

It is yet another object of the present invention to provide methods ofusing MDC and compositions comprising MDC for the augmentation of musclesoft tissue, or non-muscle soft tissue, including skin, blood vessels,adipose, nerve, skeletal muscle, smooth muscle, ligament, cartilage, andvarious organ tissues, without the need for polymer carriers or specialculture media for transplantation. Such methods include theadministration of MDC compositions by introduction into soft tissue, forexample by direct injection into tissue, or by systemic distribution ofthe compositions. Preferably, soft tissue includes non-bone bodytissues. More preferably, soft tissue includes non-striated muscle,non-bone body tissues. Most preferably, soft tissue includes non-muscle,non-bone body tissues. As used herein, augmentation refers to filling,bulking, supporting, enlarging, extending, or increasing the size ormass of body tissue.

It is another object of the present invention to provide MDC-basedtreatments for a) cosmetic or aesthetic conditions; b) gastroesophagealreflux symptoms and conditions; c) fecal and urinary incontinence; andd) skeletal and smooth muscle weakness, injury, disease, or dysfunction.

It is yet another object of the present invention to provide methods ofaugmenting bone or soft tissue, either muscle-derived soft tissue, ornon-muscle-derived soft tissue, following injury, wounding, surgeries,traumas, non-traumas, or other procedures that result in fissures,openings, depressions, wounds, and the like, in the skin or in internalsoft tissues or organs.

It is a further object of the present invention to provide MDC andcompositions comprising MDC that are modified through the use ofchemicals, growth media, and/or genetic manipulation. Such MDC andcompositions thereof comprise chemically or genetically modified cellsuseful for the production and delivery of biological compounds, and thetreatment of various diseases, conditions, injuries, or illnesses.

Additional objects and advantages afforded by the present invention willbe apparent from the detailed description and exemplificationhereinbelow.

DESCRIPTION OF THE FIGURES

The appended drawings of the figures are presented to further describethe invention and to assist in its understanding through clarificationof its various aspects.

FIGS. 1A-1F illustrate the results of soft tissue augmentation utilizinginjections of MDC compositions compared with injection of conventionalbovine collagen. For FIGS. 1A-1F, either MDC (FIGS. 1D-1F) or collagen(1A-1C) were injected into the skin of the abdominal wall. The area ofinjection was the interface of the dermis and the subcutaneousconnective tissue which is the skin. FIGS. 1A-1F show Trichrome stainingat 40× magnification following injection of either collagen or MDC intoskin. At 5 days, 2 weeks, or 4 weeks post-injection, tissue samples wereobtained and prepared for analysis. FIGS. 1A and 1D show the results ofMDC versus collagen injection into the skin at 5 days post-injection;FIGS. 1B and 1E show the results at 2 weeks post-injection; and FIGS. 1Cand 1F show the results at 4 weeks post-injection. Arrowheads in FIGS.1D-1F indicate the presence of MDC in the injected areas (deep pinkcolor). FIGS. 1A-1F demonstrate that following injection into thesubcutaneous space, MDC persisted and maintained/augmented the abdominalwall subcutaneous tissue for up to at least 4 weeks, while collagen didnot persist by 2 weeks post-injection into the skin. (Example 3).

FIGS. 2A and 2B illustrate the results of lower esophageal (FIG. 2A) andanal sphincter (FIG. 2B) soft tissue augmentation utilizing injectionsof MDC compositions. Injections were made into the gastroesophagealjunction or anal sphincter. At day 3 post-injection, tissue samples wereobtained and prepared for analysis. MDC are indicated by β-galactosidasestaining. FIG. 2A shows injected tissues at 100× magnification; FIG. 2Bshows injected tissues at 40× magnification. FIGS. 2A and 2Bdemonstrates that MDC injections maintained the lower esophagealsphincter and anal sphincter soft tissue augmentation for up to 3 daysfollowing injection

FIGS. 3A and 3B illustrate the results of bladder-ureteral junction softtissue augmentation utilizing injections of MDC compositions. Injectionswere made into the vesico-ureteral junction. At day 3 post-injection,tissue samples were obtained and prepared for analysis. MDC areindicated by β-galactosidase staining as viewed near the arrow. FIG. 3Ashows injected tissues at low (40×) magnification; FIG. 3B showsinjected tissues at high (100×) magnification. FIGS. 3A and 3Bdemonstrate that MDC injections maintained the bladder-ureteral junctionsoft tissue augmentation for up to 3 days following injection

FIGS. 4A and 4B illustrate the treatment of bladder cryoinjury utilizingsoft tissue injections of MDC compositions. Injections were made intothe bladder wall at the site of cryoinjury. At day 30 post-injection,tissue samples were obtained and prepared for staining. Arrows indicatesite of cryoinjury and MDC injection. Magnification is 100×. FIG. 4Ashows untreated cryodamaged bladder tissue. FIG. 4B shows cryodamagedbladder tissue treated with MDC injections; MDC are indicated byβ-galactosidase staining. FIGS. 4A and 4B demonstrate that MDCinjections maintained the soft tissue augmentation of the cryodamagedbladder tissue for up to 30 days following injection.

FIGS. 5A-5I illustrate cellular differentiation of MDC followinginjection into cryodamaged bladder tissue. Injections were made into thebladder wall at the site of cryoinjury, and tissue samples were obtainedand prepared for analysis at 5, 35, or 70 days post-injection. InjectedMDC are shown by staining for β-galactosidase, and undifferentiated MDCare shown by α-smooth muscle actin (α-SM actin) staining. MDC that havedifferentiated into myotubes or myofibers are shown by fast myosin heavychain (fast MyHC) staining. Arrows show fast MyHC. At day 5post-injection, multiple MDC are observed at the injection area and onlysome MDC have differentiated into myotubes, as shown by the high levelsof β-galactosidase (FIG. 5A) and α-SM actin (FIG. 5D) staining, and therelatively low levels of Fast MyHC (FIG. 5G) staining. At day 35post-injection, multiple MDC are observed at the injection area, andmany have differentiated into myotubes, as shown by the high levels ofgalactosidase staining (FIG. 5B), the decrease in α-SM actin (FIG. 5E)staining; and the increase in Fast MyHC (FIG. 5H) staining. At day 70post-injection, MDC are observed at the injection area, and almost allMDC have differentiated into myofibers, as shown by the high levels ofβ-galactosidase (FIG. 5C), the decrease in α-SM actin (FIG. 5F)staining, and the high levels of Fast MyHC (FIG. 5I) staining.Magnification is 200×. FIGS. 5A-5I demonstrate that MDC remain viableand begin differentiation for up to 70 days following injection intobladder soft tissue.

FIGS. 6A-6D illustrate the reinnervation of MDC injected into the softtissue of the urinary bladder. Innervation is indicated by acetylcholine(Ach) staining, which shows the neuromuscular junction. At day 3post-injection, few innervations are observed, as shown by Ach staining(FIG. 6A). At day 15 post injection, several innervations are observed(FIG. 6B). At day 30 post-injection, more innervations are observed(FIG. 6C). After 6 months post-injection, numerous innervations areobserved at low (100×) magnification (FIG. 6D). FIGS. 6A-6C showinjected tissue at high (200×) magnification. FIGS. 6A-6D demonstratethat MDC induce innervation for up to 6 months following injection intocryodamaged bladder tissues.

FIGS. 7A and 7B illustrate the results of soft tissue augmentation ofmyocardial smooth muscle utilizing injections of MDC compositions.Injections were made into the ventricular wall, and tissue samples wereprepared 3 days post-injection. MDC are indicated by β-galactosidasestaining. FIG. 7A shows injected tissue at low (100×) magnification;FIG. 7B shows injected tissue at high (200×) magnification.

FIGS. 8A and 8B illustrate the results of MDC injections into livertissue. Injections were made into liver tissue in the lower left lobe,and tissue samples were prepared 4 days post-injection. MDC areindicated by β-galactosidase staining. FIG. 8A shows low (100×)magnification; FIG. 8B shows high (200×) magnification.

FIGS. 9A and 9B illustrate the results of MDC injections into spleentissue. Injections were made into spleen tissue in the interior aspect,and tissue samples were prepared 4 days post-injection. MDC areindicated by β-galactosidase staining. FIG. 9A shows injected tissuesviewed by low (100×) magnification; FIG. 9B shows injected tissuesviewed by high (200×) magnification.

FIGS. 10A and 10B illustrate the results of MDC injections into spinalcord tissue. Injections were made into spinal cord tissue, and tissuesamples were prepared 4 days post-injection. MDC are indicated byβ-galactosidase staining. FIG. 10A shows injected tissues viewed by low(100×) magnification; FIG. 10B shows injected tissues viewed by high(200×) magnification. FIGS. 7A-7B, 8A-8B, 9A-9B, and 10A-10B demonstratethat MDC remain viable following injection into a variety of differenttissue types without damaging the host tissues.

FIGS. 11A-11L illustrate immunohistochemical analysis of PP1-4 and PP6cell populations from mdx mice showing expression of cell markersincluding desmin, MyoD, and myogenin (markers specific for myogeniclineages), M-cadherin (satellite cell specific marker), Bcl-2 (earlymyogenesis marker), CD34 (hematopoietic or stromal cell marker). FIGS.11A-11L demonstrate that PP1-4 and PP6 cell populations show comparablepercentage of cells expressing desmin (FIGS. 11A and 11G), MyoD (FIGS.11E and 11K), and myogenin (FIGS. 11F and 11L), while the PP6 populationshows a lower percentage of cells expressing M-cadherin (FIGS. 11D and11J), but a higher percentage of cells expressing Bcl-2 (FIGS. 11C and11I) and CD34 (FIGS. 11B and 11H), compared with the PP1-4 population.

FIGS. 12A-12I illustrate the intracellular co-localization of CD34 orBcl-2 staining with desmin staining in mouse muscle cells and vascularendothelial cells. FIG. 12A shows normal mouse muscle cells (see arrow)and vascular endothelial cells (see arrowhead) stained with anti-CD34antibodies and visualized by fluorescence microscopy. FIG. 12B shows thesame cells co-stained with desmin and collagen type IV antibodies. FIG.12C shows the same cells co-stained with Hoechst to show the nuclei.FIG. 12D shows a composite of the cells co-stained for CD34, desmin,collagen type IV, and Hoechst. FIG. 12E shows normal mouse muscle cells(see arrow) stained with anti-Bcl-2 antibodies and visualized byfluorescence microscopy. FIG. 12F shows the same cells co-stained withdesmin and collagen type IV antibodies. FIG. 12G shows the same cellsco-stained with Hoechst to show the nuclei. FIG. 12H shows a compositeof the cells co-stained for CD34, desmin, collagen type IV, and Hoechst.FIG. 12I shows satellite cells stained with anti-M-cadherin antibodies(see arrow). Cells were viewed at 40× magnification. FIGS. 12A-12Ddemonstrate the co-localization of CD34 and desmin, while FIGS. 12E-12Hdemonstrate the co-localization of Bcl-2 and desmin.

FIGS. 13A-E illustrate the morphologic changes and expression ofosteocalcin resulting from the exposure of mc13 cells to rhBMP-2. Mc13cells were incubated in growth media with or without rhBMP-2 for 6 days.FIG. 13A shows cells grown to >50% cell confluency in the absence ofrhBMP-2. FIG. 13B shows cells grown to >50% cell confluency in thepresence of 200 ng/ml rhBMP-2. FIG. 13C shows cells grown to >90% cellconfluency in the absence of rhBMP-2. FIG. 13D shows cells grown to >90%confluency in the presence of 200 ng/ml rhBMP-2. FIG. 13E shows cellsstained for osteocalcin expression (osteoblast cell marker; see arrows).Cells were viewed at 10× magnification. FIGS. 13A-13E demonstrate thatmc13 cells are capable of differentiating into osteoblasts upon exposureto rhBMP-2.

FIGS. 14A-14D illustrate the effects on the percentage of mc13 cellsexpressing desmin and alkaline phosphatase in response to rhBMP-2treatment. FIG. 14A shows desmin staining of newly isolated mc13 clones.FIG. 14B shows a phase contrast view of the same cells. FIG. 14C showsthe levels of desmin staining in mc13 cells following 6 days ofincubation in growth media with or without 200 ng/ml rhBMP-2. FIG. 14Dshows the levels of alkaline phosphate staining in PP1-4 cells and mc13cells following 6 days of incubation in growth media with or without 200ng/ml rhBMP-2. * indicates a statistically significant result (student'st-test). FIG. 14C demonstrates that a decreasing number of mc13 cellsexpress desmin in the presence of rhBMP-2, while FIG. 14D demonstratesthat an increasing number of mc13 cells express alkaline phosphatase inthe presence of rhBMP-2, suggesting decreasing myogenic characteristicsand increasing osteogenic characteristics of the cells in the presenceof rhBMP-2.

FIGS. 15A-15G illustrate the in vivo differentiation of mc13 cells intomyogenic and osteogenic lineages. Mc13 cells were stably transfectedwith a construct containing LacZ and the dystrophin gene, and introducedby intramuscular or intravenous injection into hind limbs of mdx mice.After 1.5 days, the animals were sacrificed and the hind limbmusculature was isolated for histology. FIG. 15A shows mc13 cells at theintramuscular injection site stained for LacZ. FIG. 15B shows the samecells co-stained for dystrophin. FIG. 15C shows mc13 cells in the regionof the intravenous injection stained for LacZ. FIG. 15D shows the samecells co-stained for dystrophin. In a separate experiment, mc13 cellswere transduced with adBMP-2, and 0.5-1.0×10⁶ cells were injected intohind limbs of SCID mice. After 14 days, the animals were sacrificed, andthe hind limb muscle tissues were analyzed. FIG. 15E shows radiographicanalysis of the hind limb to determine bone formation. FIG. 15F showsthe cells derived from the hind limb stained for LacZ. FIG. 15G showscells stained for dystrophin. FIGS. 15A-15D demonstrate that mc13 cellscan rescue dystrophin expression via intramuscular or intravenousdelivery. FIGS. 15E-15G demonstrate that mc13 cells are involved inectopic bone formation. Cells were viewed at the followingmagnifications: 40× (FIGS. 15A-15D), 10× (FIGS. 15F-15G)

FIGS. 16A-16E illustrate the enhancement of bone healing by rhBMP-2producing primary muscle cells. A 5 mm skull defect was created infemale SCID mice using a dental burr, and the defect was filled with acollagen sponge seeded with mc13 cells with or without adBMP-2. Theanimals were sacrificed at 14 days, inspected, and analyzedmicroscopically for indications of bone healing. FIG. 16A shows a skulltreated with mc13 cells without adBMP-2. FIG. 16B shows a skull treatedwith mc13 cells transduced with adBMP-2. FIG. 16C shows a histologicalsample of the skull treated with mc13 cells without adBMP-2 analyzed byvon Kossa staining. FIG. 16D shows a histological sample of the skulltreated with mc13 cells transduced with adBMP-2 analyzed by von Kossastaining. FIG. 16E shows a histological sample of the skull treated withthe mc13 cells transduced with adBMP-2 analyzed by hybridization with aY-chromosome specific probe to identify the injected cells (greenfluorescence shown by arrows), and stained with ethidium bromide toidentify the nuclei (indicated by red fluorescence). FIGS. 16A-16Edemonstrate that mc13 cells expressing rhBMP-2 can contribute to thehealing of bone defects.

DETAILED DESCRIPTION OF THE INVENTION

Muscle-Derived Cells and Compositions

The present invention provides MDC comprised of early progenitor cells(also termed muscle-derived progenitor cells or muscle-derived stemcells herein) that show long-term survival rates followingtransplantation into body tissues, preferably soft tissues. To obtainthe MDC of this invention, a muscle explant, preferably skeletal muscle,is obtained from an animal donor, preferably from a mammal, includinghumans. This explant serves as a structural and functional syncytiumincluding “rests” of muscle precursor cells (T. A. Partridge et al.,1978, Nature 73:306-8; B. H. Lipton et al., 1979, Science 205:1292-4).

Cells isolated from primary muscle tissue contain a mixture offibroblasts, myoblasts, adipocytes, hematopoietic, and muscle-derivedprogenitor cells. The progenitor cells of a muscle-derived populationcan be enriched using differential adherence characteristics of primarymuscle cells on collagen coated tissue flasks, such as described inpatent application U.S. Ser. No. 09/302,896 of Chancellor et al. Cellsthat are slow to adhere tend to be morphologically round, express highlevels of desmin, and have the ability to fuse and differentiate intomultinucleated myotubes (U.S. Ser. No. 09/302,896 of Chancellor et al.).A subpopulation of these cells was shown to respond to recombinant humanbone morphogenic protein 2 (rhBMP-2) in vitro by expressing increasedlevels of alkaline phosphatase, parathyroid hormone dependent3′,5′-cAMP, and osteocalcin, indicative of their ability todifferentiate through both osteogenic lineage and myogenic lineages(U.S. Ser. No. 09/302,896 of Chancellor et al.; T. Katagiri et al.,1994, J. Cell Biol. 127:1755-1766).

In accordance with the present invention, populations of rapidlyadhering MDC (PP1-4) and slowly adhering, round MDC (PP6) were isolatedand enriched from skeletal muscle explants and tested for the expressionof various markers using immunohistochemistry to determine the presenceof pluripotent cells among the slowly adhering cells (Example 1; patentapplication U.S. Ser. No. 09/302,896 of Chancellor et al.). As shown inTable 3, Example 9 herein, the PP6 cells expressed myogenic markers,including desmin, MyoD, and Myogenin. The PP6 cells also expressed c-metand MNF, two genes which are expressed at an early stage of myogenesis(J. B. Miller et al., 1999, Curr. Top. Dev. Biol. 43:191-219; see Table3). The PP6 showed a lower percentage of cells expressing M-cadherin, asatellite cell-specific marker (A. Irintchev et al., 1994, DevelopmentDynamics 199:326-337), but a higher percentage of cells expressingBcl-2, a marker limited to cells in the early stages of myogenesis (J.A. Dominov et al., 1998, J. Cell Biol. 142:537-544). The PP6 cells alsoexpressed CD34, a marker identified with human hematopoietic progenitorcells, as well as stromal cell precursors in bone marrow (R. G. Andrewset al., 1986, Blood 67:842-845; C. I. Civin et al., 1984, J. Immunol.133:157-165; L. Fina et al, 1990, Blood 75:2417-2426; P. J. Simmons etal., 1991, Blood 78:2848-2853; see Table 3). The PP6 cells alsoexpressed Flk-1, a mouse homologue of human KDR gene which was recentlyidentified as a marker of hematopoietic cells with stem cell-likecharacteristics (B. L. Ziegler et al., 1999, Science 285:1553-1558; seeTable 3). Similarly, the PP6 cells expressed Sca-1, a marker present inhematopoietic cells with stem cell-like characteristics (M. van de Rijnet al., 1989, Proc. Natl. Acad. Sci. USA 86:4634-8; M. Osawa et al.,1996, J. Immunol. 156:3207-14; see Table 3). However, the PP6 cells didnot express the CD45 or c-Kit hematopoietic stem cell markers (reviewedin L K. Ashman, 1999, Int. J. Biochem. Cell. Biol. 31:1037-51; G. A.Koretzky, 1993, FASEB J. 7:420-426; see Table 3).

Preferred in the present invention is the PP6 population ofmuscle-derived progenitor cells having the characteristics describedherein. These muscle-derived progenitor cells express the desmin, CD34,and Bcl-2 cell markers. In accordance with the present invention, thePP6 cells are isolated by the techniques described herein (Example 1) toobtain a population of muscle-derived progenitor cells that havelong-term survivability following transplantation. The PP6muscle-derived progenitor cell population comprises a significantpercentage of cells that express progenitor cell markers such as desmin,CD34, and Bcl-2. In addition, PP6 cells express the Flk-1 and Sca-1 cellmarkers, but do not express the CD45 or c-Kit markers. Preferably,greater than 95% of the PP6 cells express the desmin, Sca-1, and Flk-1markers, but do not express the CD45 or c-Kit markers. It is preferredthat the PP6 cells are utilized within about 1 day or about 24 hoursafter the last plating.

As an alternative to the pre-plating method, the MDC of the presentinvention can be isolated by fluorescence-activated cell sorting (FACS)analysis using labeled antibodies against one or more of the cellsurface markers expressed by the MDC (C. Webster et al., 1988, Exp.Cell. Res. 174:252-65; J. R. Blanton et al., 1999, Muscle Nerve22:43-50). For example, FACS analysis can be performed using labeledantibodies to directed to CD34, Flk-1, Sca-1, and/or the othercell-surface markers described herein to select a population of PP6-likecells that exhibit long-term survivability when introduced into the hosttissue. Also encompassed by the present invention is the use of one ormore fluorescence-detection labels, for example, fluorescein orrhodamine, for antibody detection of different cell marker proteins.

Muscle-Derived Cell-Based Treatments

In one embodiment of the present invention, the MDC are isolated from askeletal muscle source and introduced or transplanted into a muscle ornon-muscle soft tissue site of interest, or into bone structures.Advantageously, the MDC of the present invention are isolated andenriched to contain a large number of progenitor cells showing long-termsurvival following transplantation. In addition, the muscle-derivedprogenitor cells of this invention express a number of characteristiccell markers, such desmin, CD34, and Bcl-2. Furthermore, themuscle-derived progenitor cells of this invention express the Sca-1, andFlk-1 cell markers, but do not express the CD45 or c-Kit cell markers(see Example 1).

MDC and compositions comprising MDC of the present invention can be usedto repair, treat, or ameliorate various aesthetic or functionalconditions (e.g. defects) through the augmentation of muscle ornon-muscle soft tissues. In particular, such compositions can be used assoft-tissue bulking agents for the treatment of: 1) cosmetic andaesthetic conditions of the skin; 2) conditions of the lumen; 3)gastroesophageal reflux symptoms or conditions; 4) fecal incontinence;5) skeletal muscle weakness, disease, injury or dysfunction; and 6)smooth muscle weakness, disease, injury, or dysfunction. In addition,such MDC and compositions thereof can be used for augmenting soft tissuenot associated with injury by adding bulk to a soft tissue area,opening, depression, or void in the absence of disease or trauma, suchas for “smoothing” or removing a wrinkle. Multiple and successiveadministrations of MDC are also embraced by the present invention.

For MDC-based treatments, a skeletal muscle explant is preferablyobtained from an autologous or heterologous human or animal source. Anautologous animal or human source is more preferred. MDC compositionsare then prepared and isolated as described herein. To introduce ortransplant the MDC and/or compositions comprising the MDC according tothe present invention into a human or animal recipient, a suspension ofmononucleated muscle cells is prepared. Such suspensions containconcentrations of the muscle-derived progenitor cells of the inventionin a physiologically-acceptable carrier, excipient, or diluent. Forexample, suspensions of MDC for administering to a subject can comprise10⁸ to 10⁹ cells/ml in a sterile solution of complete medium modified tocontain the subject's serum, as an alternative to fetal bovine serum.Alternatively, MDC suspensions can be in serum-free, sterile solutions,such as cryopreservation solutions (Celox Laboratories, St. Paul,Minn.). The MDC suspensions can then be introduced e.g., via injection,into one or more sites of the donor tissue.

The described cells can be administered as a pharmaceutically orphysiologically acceptable preparation or composition containing aphysiologically acceptable carrier, excipient, or diluent, andadministered to the tissues of the recipient organism of interest,including humans and non-human animals. The MDC-containing compositioncan be prepared by resuspending the cells in a suitable liquid orsolution such as sterile physiological saline or other physiologicallyacceptable injectable aqueous liquids. The amounts of the components tobe used in such compositions can be routinely determined by those havingskill in the art.

The MDC or compositions thereof can be administered by placement of theMDC suspensions onto absorbent or adherent material, i.e., a collagensponge matrix, and insertion of the MDC-containing material into or ontothe site of interest. Alternatively, the MDC can be administered byparenteral routes of injection, including subcutaneous, intravenous,intramuscular, and intrasternal. Other modes of administration include,but are not limited to, intranasal, intrathecal, intracutaneous,percutaneous, enteral, and sublingual. In one embodiment of the presentinvention, administration of the MDC can be mediated by endoscopicsurgery.

For injectable administration, the composition is in sterile solution orsuspension or can be resuspended in pharmaceutically- andphysiologically-acceptable aqueous or oleaginous vehicles, which maycontain preservatives, stabilizers, and material for rendering thesolution or suspension isotonic with body fluids (i.e. blood) of therecipient. Non-limiting examples of excipients suitable for use includewater, phosphate buffered saline, pH 7.4, 0.15 M aqueous sodium chloridesolution, dextrose, glycerol, dilute ethanol, and the like, and mixturesthereof. Illustrative stabilizers are polyethylene glycol, proteins,saccharides, amino acids, inorganic acids, and organic acids, which maybe used either on their own or as admixtures. The amounts or quantities,as well as the routes of administration used, are determined on anindividual basis, and correspond to the amounts used in similar types ofapplications or indications known to those of skill in the art.

To optimize transplant success, the closest possible immunological matchbetween donor and recipient is desired. If an autologous source is notavailable, donor and recipient Class I and Class II histocompatibilityantigens can 0 be analyzed to determine the closest match available.This minimizes or eliminates immune rejection and reduces the need forimmunosuppressive or immunomodulatory therapy. If required,immunosuppressive or immunomodulatory therapy can be started before,during, and/or after the transplant procedure. For example, cyclosporinA or other immunosuppressive drugs can be administered to the transplantrecipient. Immunological tolerance may also be induced prior totransplantation by alternative methods known in the art (D. J Watt etal., 1984, Clin. Exp. Immunol. 55:419; D. Faustman et al., 1991, Science252:1701).

Consistent with the present invention, the MDC can be administered tobody tissues, including bone, epithelial tissue (i.e., skin, lumen,etc.), connective tissue (i.e., adipose, cartilage, ligament, lymph,etc.), muscle tissue (i.e., skeletal/striated or smooth muscle), andvarious organ tissues such as those organs that are associated with thedigestive system (i.e., mouth, tongue, esophagus, stomach, liver,pancreas, gall bladder, intestine, anus, etc.), cardiovascular system(i.e., heart, veins, arteries, capillaries, etc.), respiratory system(i.e., lungs, trachea, etc.), reproductive system (i.e., vas deferens,scrotum, testes, penis, fallopian tubes, vagina, clitoris, uterus,breasts, ovaries, vulva, etc.), urological system (i.e.; bladder,urethra, ureter, kidneys, etc.), and nervous system (i.e., brain, spinalcord, nerves, etc.).

The number of cells in an MDC suspension and the mode of administrationmay vary depending on the site and condition being treated. Asnon-limiting examples, in accordance with the present invention, about1-1.5×10⁶ MDC are injected for the treatment of an approximately 8 mmdiameter region of cryodamage in bladder smooth muscle tissue (seeExample 6), while about 0.5-1.0×10⁶ MDC are administered via a collagensponge matrix for the treatment of an approximately 5 mm region of skulldefect (see Example 9). Consistent with the Examples disclosed herein, askilled practitioner can modulate the amounts and methods of MDC-basedtreatments according to requirements, limitations, and/or optimizationsdetermined for each case.

Dermatological conditions: The MDC and compositions thereof according tothe present invention have marked utility as materials for soft tissueaugmentation in cosmetic procedures, e.g., plastic surgery or anti-agingprocedures. Specifically, such MDC and MDC-containing compositions canbe used to treat various dermatological conditions in a human or animalsubject, including, but not limited to, wounds, wrinkles, rhytids,cutaneous depressions of non-traumatic origin, stretch marks, depressedscars, scaring from acne vulgaris, and hypoplasia of the lip. Morespecifically, the MDC and compositions of the present invention can beused to treat wrinkles, rhytids, or cutaneous depressions of the face,and especially, the region surrounding the eye(s). To treatdermatological conditions, the MDC are prepared as disclosed herein andthen administered, e.g. via injection, to the skin, subcutaneously orintradermally, to fill, bulk up, or repair the defect. The number of MDCintroduced is modulated to repair deep cutaneous depressions or defects,as well as superficial surface depressions or defects, as required. Forexample, about 1-1.5×10⁶ MDC are utilized for the augmentation of anapproximately 5 mm region of the skin (see Example 3).

Conditions of the lumen: In another embodiment, the MDC and compositionsthereof according to the present invention have further utility astreatments for conditions of the lumen in an animal or mammal subject,including humans. Specifically, the muscle-derived progenitor cells areused for completely or partially blocking, enhancing, enlarging,sealing, repairing, bulking, or filling various biological lumens orvoids within the body. Lumens include, without limitation, bloodvessels, intestine, stomach, esophagus, urethra, vagina, Fallopiantubes, vas deferens, and trachea. Voids may include, without limitation,various tissue wounds (i.e., loss of muscle and soft tissue bulk due totrauma; destruction of soft tissue due to penetrating projectiles suchas a stab wound or bullet wound; loss of soft tissue from disease ortissue death due to surgical removal of the tissue including loss ofbreast tissue following a mastectomy for breast cancer or loss of muscletissue following surgery to treat sarcoma, etc.), lesions, fissures,diverticulae, cysts, fistulae, aneurysms, and other undesirable orunwanted depressions or openings that may exist within the body of ananimal or mammal, including humans. For the treatment of conditions ofthe lumen, the MDC are prepared as disclosed herein and thenadministered, e.g. via injection or intravenous delivery, to the lumenaltissue to fill or repair the void. The number of MDC introduced ismodulated to repair large or small voids in a soft tissue environment,as required.

Conditions of the sphincter: The MDC and compositions thereof accordingto the present invention can also be used for the treatment of asphincter injury, weakness, disease, or dysfunction in an animal ormammal, including humans. In particular, the MDC are used to augmenttissues of the esophageal, anal, cardiac, pyloric, and urinarysphincters. More specifically, the present invention provides softtissue augmentation treatments for gastroesophageal reflux symptoms, andurinary and fecal incontinence. For the treatment of sphincter defects,the MDC are prepared as described herein and then administered to thesphincter tissue, e.g. via injection, to provide additional bulk,filler, or support. The number of MDC introduced is modulated to providevarying amounts of bulking material as required. For example, about1-1.5×10⁶ MDC are used to provide augmentation for an approximately 5 mmregion of the gastroesophageal junction or an approximately 5-10 mmregion of the anal sphincter (see Example 4).

Muscle augmentation and contractility: In yet another embodiment of thepresent invention, the MDC and compositions thereof are used for thetreatment of muscle conditions in a human or animal subject. Inparticular, the MDC can be used to augment the skeletal or smoothmuscles to treat weakness or dysfunction caused by injury, disease,inactivity, or anoxia- or surgery-induced trauma. More specifically, thepresent invention provides treatments for skeletal muscle weakness ordysfunction, such as a sports-related injury. The present invention alsoprovides treatments for smooth muscle disease or dysfunction, such asheart failure, or injury associated with myocardial infarction.

For muscle augmentation or treatment of muscle-related conditions, theMDC are prepared as described above and are administered, e.g. viainjection, into muscle tissue to provide additional bulk, filler, orsupport. As is appreciated by the skilled practitioner, the number ofMDC introduced is modulated to provide varying amounts of bulkingmaterial, as needed or required. For example, about 1-1.5×10⁶ MDC areinjected for the augmentation of an approximately 5 mm region of hearttissue (see Example 7).

In addition, the MDC and compositions thereof can be used to affectcontractility in smooth muscle tissue, such as gastrointestinal tissue,esophageal tissue, and bladder tissue, as example. Indeed, musclecontractility was seen to be restored in cryodamaged bladder tissueafter the introduction of muscle-derived progenitor cells, i.e., MDC, asdemonstrated in Example 6. Thus, the present invention also embraces theuse of MDC of the invention in restoring muscle contraction, and/orameliorating or overcoming smooth muscle contractility problems, suchdecreased gastrointestinal motility, including the esophagus, stomachand intestine smooth muscle. A specific, yet nonlimiting example of acondition that the MDC of the invention can improve, reduce, or correctis gastroparesis, i.e., poor motility and emptying of the stomach.

Genetically Engineered Muscle-Derived Cells

In another aspect of the present invention, the MDC of this inventionmay be genetically engineered to contain a nucleic acid sequence(s)encoding one or more active biomolecules, and to express thesebiomolecules, including proteins, polypeptides, peptides, hormones,metabolites, drugs, enzymes, and the like. Such MDC may behistocompatible (autologous) or nonhistocompatible (allogeneic) to therecipient, including humans. These cells can serve as long-term localdelivery systems for a variety of treatments, for example, for thetreatment of such diseases and pathologies as cancer, transplantrejection, and the regeneration of muscle and nerve tissues, diabetes,liver failure, renal failure, neural defects and diseases such asParkinson's disease, and to deliver a gene product to a site of tissueaugmentation, or void filling, such as a therapeutic agent, as describedherein.

Preferred in the present invention are autologous muscle-derivedprogenitor cells, which will not be recognized as foreign to therecipient. In this regard, the MDC used for cell-mediated gene transferor delivery will desirably be matched vis-à-vis the majorhistocompatibility locus (MHC or HLA in humans). Such MHC or HLA matchedcells may be autologous. Alternatively, the cells may be from a personhaving the same or a similar MHC or HLA antigen profile. The patient mayalso be tolerized to the allogeneic MHC antigens. The present inventionalso encompasses the use of cells lacking MHC Class I and/or IIantigens, such as described in U.S. Pat. No. 5,538,722.

The MDC may be genetically engineered by a variety of moleculartechniques and methods known to those having skill in the art, forexample, transfection, infection, or transduction. Transduction as usedherein commonly refers to cells that have been genetically engineered tocontain a foreign or heterologous gene via the introduction of a viralor non-viral vector into the cells. Transfection more commonly refers tocells that have been genetically engineered to contain a foreign geneharbored in a plasmid, or non-viral vector. MDC can be transfected ortransduced by different vectors and thus can serve as gene deliveryvehicles to transfer the expressed products into muscle.

Although viral vectors are preferred, those having skill in the art willappreciate that the genetic engineering of cells to contain nucleic acidsequences encoding desired proteins or polypeptides, cytokines, and thelike, may be carried out by methods known in the art, for example, asdescribed in U.S. Pat. No. 5,538,722, including fusion, transfection,lipofection mediated by the use of liposomes, electroporation,precipitation with DEAE-Dextran or calcium phosphate, particlebombardment (biolistics) with nucleic acid-coated particles (e.g., goldparticles), microinjection, and the like.

Vectors for introducing heterologous (i.e., foreign) nucleic acid (DNAor RNA) into muscle cells for the expression of bioactive products arewell known in the art. Such vectors possess a promoter sequence,preferably, a promoter that is cell-specific and placed upstream of thesequence to be expressed. The vectors may also contain, optionally, oneor more expressible marker genes for expression as an indication ofsuccessful transfection and expression of the nucleic acid sequencescontained in the vector.

Illustrative examples of vehicles or vector constructs for transfectionor infection of the muscle-derived cells of the present inventioninclude replication-defective viral vectors, DNA virus or RNA virus(retrovirus) vectors, such as adenovirus, herpes simplex virus andadeno-associated viral vectors. Adeno-associated virus vectors aresingle stranded and allow the efficient delivery of multiple copies ofnucleic acid to the cell's nucleus. Preferred are adenovirus vectors.The vectors will normally be substantially free of any prokaryotic DNAand may comprise a number of different functional nucleic acidsequences. Examples of such functional sequences include polynucleotide,e.g., DNA or RNA, sequences comprising transcriptional and translationalinitiation and termination regulatory sequences, including promoters(e.g., strong promoters, inducible promoters, and the like) andenhancers which are active in muscle cells.

Also included as part of the functional sequences is an open readingframe (polynucleotide sequence) encoding a protein of interest; flankingsequences may also be included for site-directed integration. In somesituations, the 5′-flanking sequence will allow homologousrecombination, thus changing the nature of the transcriptionalinitiation region, so as to provide for inducible or noninducibletranscription to increase or decrease the level of transcription, as anexample.

In general, the nucleic acid sequence desired to be expressed by themuscle-derived progenitor cell is that of a structural gene, or afunctional fragment, segment or portion of the gene, that isheterologous to the muscle-derived progenitor cell and encodes a desiredprotein or polypeptide product, for example. The encoded and expressedproduct may be intracellular, i.e., retained in the cytoplasm, nucleus,or an organelle of a cell, or may be secreted by the cell. Forsecretion, the natural signal sequence present in the structural genemay be retained, or a signal sequence that is not naturally present inthe structural gene may be used. When the polypeptide or peptide is afragment of a protein that is larger, a signal sequence may be providedso that, upon secretion and processing at the processing site, thedesired protein will have the natural sequence. Examples of genes ofinterest for use in accordance with the present invention include genesencoding cell growth factors, cell differentiation factors, cellsignaling factors and programmed cell death factors. Specific examplesinclude, but are not limited to, genes encoding BMP-2 (rhBMP-2), IL-1Ra, Factor IX, and connexin 43.

As mentioned above, a marker may be present for selection of cellscontaining the vector construct. The marker may be an inducible ornon-inducible gene and will generally allow for positive selection underinduction, or without induction, respectively. Examples of commonly-usedmarker genes include neomycin, dihydrofolate reductase, glutaminesynthetase, and the like.

The vector employed will generally also include an origin of replicationand other genes that are necessary for replication in the host cells, asroutinely employed by those having skill in the art. As an example, thereplication system comprising the origin of replication and any proteinsassociated with replication encoded by a particular virus may beincluded as part of the construct. The replication system must beselected so that the genes encoding products necessary for replicationdo not ultimately transform the muscle-derived cells. Such replicationsystems are represented by replication-defective adenovirus constructedas described, for example, by G. Acsadi et al., 1994, Hum. Mol. Genet.3:579-584, and by Epstein-Barr virus. Examples of replication defectivevectors, particularly, retroviral vectors that are replicationdefective, are BAG, described by Price et al., 1987, Proc. Natl. Acad.Sci. USA, 84:156; and Sanes et al., 1986, EMBO J., 5:3133. It will beunderstood that the final gene construct may contain one or more genesof interest, for example, a gene encoding a bioactive metabolicmolecule. In addition, cDNA, synthetically produced DNA or chromosomalDNA may be employed utilizing methods and protocols known and practicedby those having skill in the art.

If desired, infectious replication-defective viral vectors may be usedto genetically engineer the cells prior to in vivo injection of thecells. In this regard, the vectors may be introduced into retroviralproducer cells for amphotrophic packaging. The natural expansion ofmuscle-derived progenitor cells into adjacent regions obviates a largenumber of injections into or at the site(s) of interest.

In another aspect, the present invention provides ex vivo gene deliveryto cells and tissues of a recipient mammalian host, including humans,through the use of MDC, e.g., early progenitor muscle cells, that havebeen virally transduced using an adenoviral vector engineered to containa heterologous gene encoding a desired gene product. Such an ex vivoapproach provides the advantage of efficient viral gene transfer, whichis superior to direct gene transfer approaches. The ex vivo procedureinvolves the use of the muscle-derived progenitor cells from isolatedcells of muscle tissue. The muscle biopsy that will serve as the sourceof muscle-derived progenitor cells can be obtained from an injury siteor from another area that may be more easily obtainable from theclinical surgeon.

It will be appreciated that in accordance with the present invention,clonal isolates can be derived from the population of muscle-derivedprogenitor cells (i.e., PP6 cells) using various procedures known in theart, for example, limiting dilution plating in tissue culture medium.Clonal isolates comprise genetically identical cells that originate froma single, solitary cell. In addition, clonal isolates can be derivedusing FACS analysis as described above, followed by limiting dilution toachieve a single cell per well to establish a clonally isolated cellline. An example of a clonal isolate derived from the PP6 cellpopulation is mc13, which is described in Example 9. Preferably, MDCclonal isolates are utilized in the present methods, as well as forgenetic engineering for the expression of one or more bioactivemolecules, or in gene replacement therapies.

The MDC are first infected with engineered viral vectors containing atleast one heterologous gene encoding a desired gene product, suspendedin a physiologically acceptable carrier or excipient, such as saline orphosphate buffered saline, and then administered to an appropriate sitein the host. Consistent with the present invention, the MDC can beadministered to body tissues, including bone, epithelial tissue,connective tissue, muscle tissue, and various organ tissues such asthose organs that are associated with the digestive system,cardiovascular system, respiratory system, reproductive system,urological system, and nervous system, as described above. The desiredgene product is expressed by the injected cells, which thus introducethe gene product into the host. The introduced and expressed geneproducts can thereby be utilized to treat, repair, or ameliorate theinjury, dysfunction, or disease, due to their being expressed over longtime periods by the MDC of the invention, having long-term survival inthe host.

In animal model studies of myoblast-mediated gene therapy, implantationof 10⁶ myoblasts per 100 mg muscle was required for partial correctionof muscle enzyme defects (see, J. E. Morgan et al., 1988, J. Neural.Sci. 86:137; T. A. Partridge et al., 1989, Nature 337:176).Extrapolating from this data, approximately 10¹² MDC suspended in aphysiologically compatible medium can be implanted into muscle tissuefor gene therapy for a 70 kg human. This number of MDC of the inventioncan be produced from a single 100 mg skeletal muscle biopsy from a humansource (see below). For the treatment of a specific injury site, aninjection of genetically engineered MDC into a given tissue or site ofinjury comprises a therapeutically effective amount of cells in solutionor suspension, preferably, about 10⁵ to 10⁶ cells per cm³ of tissue tobe treated, in a physiologically acceptable medium.

EXAMPLES Example 1 MDC Enrichment, Isolation and Analysis

Enrichment and isolation of MDC: MDC were prepared as described (patentapplication U.S. Ser. No. 09/302,896 of Chancellor et al.). Muscleexplants were obtained from the hind limbs of a number of sources,namely from 3-week-old mdx (dystrophic) mice (C57BL/10ScSn mdx/mdx,Jackson Laboratories), 4-6 week-old normal female SD (Sprague Dawley)rats, or SCID (severe combined immune deficiency) mice. The muscletissue from each of the animal sources was dissected to remove any bonesand minced into a slurry. The slurry was then digested by 1 hour serialincubations with 0.2% type XI collagenase, dispase (grade II, 240 unit),and 0.1% trypsin at 37° C. The resulting cell suspension was passedthrough 18, 20, and 22 gauge needles and centrifuged at 3000 rpm for 5minutes. Subsequently, cells were suspended in growth medium (DMEMsupplemented with 10% fetal bovine serum, 10% horse serum, 0.5% chickembryo extract, and 2% penicillin/streptomycin). Cells were thenpreplated in collagen-coated flasks (patent application U.S. Ser. No.09/302,896 of Chancellor et al.). After approximately 1 hour, thesupernatant was removed from the flask and re-plated into a freshcollagen-coated flask. The cells which adhered rapidly within this 1hour incubation were mostly fibroblasts (Z. Qu et al., supra;application U.S. Ser. No. 09/302,896 of Chancellor et al.). Thesupernatant was removed and re-plated after 30-40% of the cells hadadhered to each flask. After approximately 5-6 serial platings, theculture was enriched with small, round cells, designated as PP6 cells,which were isolated from the starting cell population and used infurther studies. The adherent cells isolated in the early platings werepooled together and designated as PP1-4 cells.

The mdx PP1-4, mdx PP6, normal PP6, and fibroblast cell populations wereexamined by immunohistochemical analysis for the expression of cellmarkers. The results of this analysis are shown in Table 1.

TABLE 1 Table 1: Cell markers expressed in PP1-4 and PP6 cellpopulations. mdx PP1-4 cells mdx PP6 cells nor PP6 cells fibroblastsdesmin +/− + + − CD34 − + + − Bcl-2 (−) + + − Flk-1 na + + − Sca-1na + + − M-cadherin −/+ −/+ −/+ − MyoD −/+ +/− +/− − myogenin −/+ +/−+/− − Mdx PP1-4, mdx PP6, normal PP6, and fibroblast cells were derivedby preplating technique and examined by immunohistochemical analysis.“−” indicates less than 2% of the cells showed expression; “(−)”; “−/+”indicates 5-50% of the cells showed expression; “+/−” indicates ~40-80%of the cells showed expression; “+” indicates that >95% of the cellsshowed expression; “nor” indicates normal cells; “na” indicates that theimmunohistochemical data is not available.It is noted that both mdx and normal mice showed identical distributionof all of the cell markers tested in this assay. Thus, the presence ofthe mdx mutation does not affect the cell marker expression of theisolated PP6 muscle-cell derived population.

MDC were grown in proliferation medium containing DMEM (Dulbecco'sModified Eagle Medium) with 10% FBS (fetal bovine serum), 10% HS (horseserum), 0.5% chick embryo extract, and 1% penicillin/streptomycin, orfusion medium containing DMEM supplemented with 2% fetal bovine serumand 1% antibiotic solution. All media supplies were purchased throughGibco Laboratories (Grand Island, N.Y.).

Example 2 MDC Vectors and Transfection

Retrovirus and adenovirus vectors: The MFG-NB (N. Ferry et al., 1991,Proc. Natl. Acad. Sci. USA 88:8377-81) retroviral vector was used forthe MDC experiments. This vector contains a modified LacZ gene(NLS-LacZ) that includes a nuclear-localization sequence cloned from thesimian virus (SV40) large T antigen transcribed from the long terminalrepeat (LTR). The retroviral stock was grown and prepared as previouslydescribed (J. C. van Deutekom et al., 1998, Neuromuscul. Disord.8:135-48). The retrovirus was titered to 1×10⁷-1×10⁹ cfu/ml.

An adenovirus vector was also used. This vector contained the LacZ geneunder the control of the human cytomegalovirus (HuCMV) promoter (J.Huard et al., 1994, Hum Gene Ther 5:949-58). The E1-E3 deletedrecombinant adenovirus was obtained through Dr. I. Kovesdi (Gene VecInc., Rockville, Md.).

Viral transduction of MDC: For viral transduction, MDC were plated at adensity of 1-1.5×10⁶ in T 75 flasks. PP6 MDC were washed in HBSS (Hank'sBalanced Salt Solution) and incubated with either retrovirus(1×10⁷-1×10⁹ cfu/ml) or adenovirus (1×10⁹ cfu/ml) suspensions in 5 ml ofDMEM containing 8 μg/ml Polybrenen™ (Abbott Laboratories, Chicago, Ill.)for 4 h at 37° C. Virally transduced MDC were grown in 10 ml ofproliferation medium for 24 h at 37° C. MDC were then rinsed with HBSSand enzymatically digested with 0.25% trypsin for 1 minute. The treated,virally transduced MDC were centrifuged for 5 minutes at 3,500 rpm, andthe pellet was resuspended in 20 μl of HBSS.

Example 3 Soft Tissue Augmentation of the Skin

MDC and collagen injection: SD rats were prepared for surgery byanesthetizing with halothane using standard methods, and washing thesurgical site with Betadine® solution. The skin of the lower abdomen wasinjected with either 10 microliters (μl) of a MDC suspension in HBSS(approximately 1-1.5×10⁶ cells), 10 μl of commercially available bovinecollagen (Contigen™; C. R. Bard, Covington, Ga.), or 10 μl of sterilesaline using a Hamilton microsyringe. At 5 days, 2 weeks and 4 weekspost-injection, the area surrounding each injection site was excised,prepared for histochemical analysis, examined microscopically, andphotographed. Histochemical analysis included hematoxylin, eosin, ortrichrome staining.

The results demonstrate that MDC were viable for up to at least 4 weeksfollowing injection into skin tissue, with no evidence of inflammationof the tissue at the injection site (FIGS. 1D-1F). In contrast, collagenwas not visible at 2 weeks following injection into skin tissue (FIGS.1B and 1C). Thus, MDC compositions can be used as skin augmentationmaterials for use, for example, in cosmetic and aesthetic applicationsor surgery. This is an unexpected finding, since it was previouslybelieved that transplanted muscle cells needed surrounding host musclefibers with which to attach in order to survive. The survival of the MDCof the present invention following injection into non-muscle tissue isfurther demonstrated in Examples 8 and 9.

Example 4 Soft Tissue Augmentation of the Gastroesophageal Junction andAnal Sphincter

SD rats were prepared for surgery as described above. A midline abdomenincision was made to expose the gastroesophageal junction and analsphincter. The soft tissue was injected with 10 μl of a suspension ofmuscle-derived progenitor cells of in HBSS (1-1.5×10⁶ cells) using aHamilton microsyringe. At day 3 post-injection, the area surroundingeach injection site was excised, prepared for histochemical analysis,stained for β-galactosidase to determine the location and viability ofthe cells carrying the LacZ marker, examined microscopically, andphotographed. Results of these experiments demonstrate that MDCcompositions can be used as esophageal and anal sphincter bulkingmaterials (FIGS. 2A and 2B) for the treatment of gastroesophageal refluxor fecal incontinence symptoms or conditions.

Example 5 Soft Tissue Augmentation of the Vesico-Ureteral Function

SD rats were prepared for surgery as described above. A midline abdomenincision was made to expose the ureteral-bladder (vesico-ureteral)junction. The tissue was injected with 10 μl of MDC suspension in HBSS(1-1.5×10⁶ cells) using a Hamilton microsyringe. At 3 dayspost-injection, the area surrounding each injection site was excised,prepared for histological analysis, stained for β-galactosidase todetermine the location and viability of the cells carrying the LacZmarker, examined microscopically, and photographed. These resultsdemonstrate that MDC-based compositions can be used as utereral-bladderaugmentation materials (FIGS. 3A and 3B) for the treatment ofvesico-utereal reflux symptoms or conditions.

Example 6 MDC Treatment of Cryodamaged Bladder Tissue

Cryoinjury and MDC transplantation: SD rats were prepared for surgery asdescribed above. A low midline incision was made to expose the bladderand urethra. The bladder was then filled with 1 ml saline. Cryodamagewas performed with an 8 mm diameter aluminum rod chilled on dry ice. Thechilled probe was placed against one side of the bladder wall for 15 or30 seconds (referred to as “mild” or “severe” damage, respectively).Immediately following cryoinjury, one severe damage group was injectedwith muscle-derived cells of the invention (1-1.5×10⁶ of cells in 15 μlHBSS), while a control severe damage group was injected with HBSS (150)(n=3 per group). One week following cryoinjury, the other mild andsevere damage groups were injected with an MDC suspension in 50 μl HBSS(2-3×10⁶ cells), while control mild and severe damage groups wereinjected with 50 μl HBSS (n=4 per group). For each group, injectionswere made into the center of the injured region using a 30-gauge needleand a Hamilton microsyringe.

Immunohistochemical staining for smooth muscle actin (α-SM actin): Toprepare samples for immunohistochemical analysis, tissues or cellsamples were fixed in cold acetone at −20° C. for 2 minutes, and blockedwith 5% HS for 1 hour. The samples were incubated overnight at roomtemperature in a humidity chamber with mouse monoclonal anti-smoothmuscle actin primary antibodies (Cat. #F-3777; Sigma Chemical Co., St.Louis, Mo.) (1:400 dilution in PBS pH 7.4). The samples were then washed3 times with PBS, and incubated with anti-mouse IgG secondary antibodiesconjugated with the Cy3 fluorochrome (Sigma Chemical Co.) (1:200dilution in PBS pH 7.4).

Immunohistochemical staining for fast myosin heavy chain (Fast MyHC):Tissues or cell samples were fixed in cold acetone at −20° C. for 2minutes and blocked with 5% HS for 1 hour. The samples were thenincubated overnight at room temperature in a humidity chamber with mousemonoclonal anti-skeletal myosin (fast) primary antibodies (Cat. #M-4276;Sigma Chemical Co.) (1:400 dilution in PBS pH 7.4). The samples werethen washed 3 times with PBS, and incubated with Cy3 conjugatedanti-mouse IgG secondary antibodies (Sigma Chemical Co.) (1:200 dilutionin PBS pH 7.4).

Cell culture: Muscle derived progenitor cells as prepared in Example 1were plated in 35 mm collagen-coated dishes in proliferation medium.After 24 hours, the proliferation medium was replaced with fusionmedium. The cells were maintained in fusion medium with daily mediumchanges until the MDC differentiated into myotubes.

Contractility studies: Two weeks after the MDC injection, the animalswere euthanized and used to prepare bladder strips. Two strips wereprepared from each bladder, and both strips were cut to extend along thecircumference of the bladder. The bladder strips were mounted in atissue bath and subjected to neural contractions (20 Hz, 10 and 80shocks), which were recorded and analyzed as described below.

Tissue harvest and histology: SD rats were euthanized and samples of thetissue surrounding the injection site were removed. The samples wereflash frozen using 2-methylbutane pre-cooled in liquid nitrogen.Histochemical analysis of the samples included hematoxylin and eosinstaining. The samples were stained, examined microscopically, andphotographed. Each cryostat section measured 10 μm in thickness.

Electrostimulation of bladder smooth muscle tissue: The animal waseuthanized and the bladder was quickly removed. Two strips covering thecircumference of the bladder wall were obtained from each bladder. Thestrips were mounted in 5 ml organ baths containing Kreb's solution (113mmol/l NaCl, 4.7 mmol/l KCl, 1.25 mmol/l CaCl₂, 1.2 mmol/l MgSO₄, 25mmol/l NaHCO₃, 1.2 mmol/l KH₂PO₄, and 11.5 mmol/l glucose) aerated with95% O₂ and 5% CO₂. The initial tension was set to 10 mN, and isometriccontractions were measured with strain-gauge transducers coupled with aTBM4 strain gauge amplifier (World Precision Instruments). Contractionmeasurements were compiled using a data acquisition program (Windaq,DATAQ Instruments, Inc., Akron, Ohio). The sampling rate per channel wasset to 100 Hz. The amplitude of the contractions was computed using acalculation program (WindaqEx, DATAQ Instruments, Inc.). Following a 20minutes equilibration period, electrical field stimuli were appliedthrough two platinum wire electrodes separated by 4 cm at the top andthe bottom of the organ bath. The temperature was maintained at 37° C.throughout the experiment.

Chemical stimulation of bladder smooth muscle tissue: The bladder stripswere stimulated with square wave pulses of 0.25 msec duration withmaximal voltage (100 V) and a frequency response curve constructed using10 or 80 shocks at 1, 2, 5, 10, 20, or 40 Hz. Followingelectrostimulation, 5, 10, or 20 μM carbachol was added to the bladderstrips to induce contractions. In parallel experiments, 1 μM atropinewas added, electrostimulation was applied as above, and 50 μM methyleneATP was added to induce contractions.

Staining for innervation: Acetylcholine (Ach) staining was used toassess the reinnervation of MDC in smooth muscle: Ach is a stain for theneuromuscular junction that indicates the presence of nerve endings.Following MDC injection, tissue was excised at day 3, 15, 30, or after 6months, stained for Ach, observed by microscopy, and photographed.

Statistical analysis: Values are reported as means±standard deviations.A “P” value of less than 0.05 was considered statistically significant.Student's test was used.

MDC differentiation: Muscle derived progenitor cells as prepared inExample 1 were evaluated for cellular differentiation. Alpha-SM actin isthe earliest known marker for the smooth muscle cell phenotype (K. M.McHugh, 1995, Dev. Dyn. 204:278-90), and the main marker of themyofibroblastic phenotype (I. Darby et al., 1990, Lab. Invest. 63:21-9).During muscle cell differentiation, expression of α-SM actin decreases,while fast MyHC expression increases. Histochemical analysis ofMDC-treated bladder tissues utilizing α-SM actin and fast MyHC markersdemonstrates the differentiation of MDC following injection into site ofcryoinjury. At day 5 following injection into cryodamaged bladdertissue, several MDC (at least 20%) show α-SM actin staining (FIG. 5B),indicating that the cells are still undifferentiated. After 6 monthsfollowing injection, however, virtually all MDC have differentiated intomyotubes or myofibers, as shown by an decrease in α-SM actin staining(FIG. 5F), with a concomitant increase in fast MyHC staining (FIG. 5I).

Muscle reinnervation: Because acetylcholine (Ach) is present at theneuromuscular junction, it can serve as an indicator of muscleinnervation. Histochemical analysis of MDC-treated bladder tissuesutilizing the Ach marker demonstrates the reinnervation of the MDCfollowing injection into sites of cryodamage. At day 3 followinginjection into cryodamaged bladder tissue the injected MDC show minimalinnervation, as indicated by relatively low levels of Ach staining (FIG.6A). At day 15 post-injection, increased levels of innervation areobserved, as indicated by increased levels of Ach staining (FIG. 6B). Atday 30 post-injection, still more Ach staining is observed (FIG. 6C),indicative of further increases in innervation. At 6 months followinginjection, extensive innervation is observed, as indicated bysubstantial Ach staining throughout the MDC injected area viewed at lowmagnification (FIG. 6D). These results indicate that the pelvic nerve isgrowing into the MDC injected area of the bladder, and suggest that theMDC can improve the contractility and function of the injected tissue.

Contractility physiology studies: To determine whether injected MDCimproved the function of the treated bladder tissues, severalcontractility studies were completed (see above). Table 2 presents thedata showing the contractile parameters of bladder muscle followingcryoinjury with or without MDC injections.

TABLE 2 Table 2: Contractile parameters of bladder muscle followingcryoinjury. Group Contraction amplitude (mN/mgVelocity(contraction)(mN/s) No. of No. 20 Hz/10shocks 20 Hz/80shoc 20Hz/10shocks 20 Hz/80shock specimens 1 Sham 0.375 ± 0.24 0.697 ± 0.4618.08 ± 8.15 15.56 ± 8.39 6 MDC 0.368 ± 0.26 0.812 ± 0.31 16.23 ± 10.316.38 ± 7.54 6 2 Sham 0.427 ± 0.17 0.966 ± 0.31 22.96 ± 8.93 24.56 ±5.03 8 MDC 0.539 ± 0.24 1.161 ± 0.55 27.86 ± 14.08 30.59 ± 13.05 8 3Sham 0.389 ± 0.14* 0.708 ± 0.26** 25.70 ± 5.87 24.24 ± 6.38 8 MDC 0.564± 0.16* 1.139 ± 0.29** 30.59 ± 17.8 29.31 ± 15.3 8 4 Normal 0.927 ± 0.231.748 ± 0.52 34.23 ± 8.82 29.05 ± 7.06 6 *p < 0.05, **p < 0.01 Valuesare means ± standard deviations. For statistical analysis, Student'stest was performed for control and MDC injection groups. Group No. 1:severe damage group with immediate MDC injections following cryoinjury.Group No. 2: mild damage group with MDC injections one week followingcryodamage. Group No. 3: severe damage group with MDC injections oneweek following cryodamage. Group No. 4: normal bladder tissue.

The severe damage group injected with MDC immediately followingcryoinjury (Group 1) showed similar contractility as the control (sham)group (compare contractility levels shown in sham and MDC rows in Group1, Table 2). However, the severe damage group injected with MDC one weekfollowing the cryodamage (Group 3) showed increased contractionamplitude (145% and 161% of the control bladder at 20 Hz/10 shocks and20 Hz/80 shocks, respectively) compared with the control group (comparecontractile amplitude levels shown in sham and MDC rows indicated withasterisks in Table 2). Similarly, the severe damage group injected withMDC one week following the cryodamage (Group 3) showed increasedcontraction velocity (119% and 121% of that of the control strip at 20Hz/10 shocks and 20 Hz/80 shocks, respectively) compared with thecontrol group (compare contractile velocity values in sham and MDC rowsin Group 3, Table 2). The mild damage group injected with MDC one weekfollowing the cryodamage (Group 2) also showed increased contractionamplitude and velocity compared to the control group (comparecontractility levels shown in sham and MDC rows in Group 2, Table 2).The results of these studies show that MDC injections can restorecontractility to cryodamaged bladder muscle tissue, and indicate thatMDC-based compositions can be utilized for the treatment of urinaryincontinence.

Example 7 Soft Tissue Augmentation of the Myocardium

SD rats were prepared for surgery as described above. A thoracicincision was made to expose the heart. The ventricular wall was injectedwith 10 μl of MDC suspension in HBSS (1-1.5×10⁶ cells) using a Hamiltonmicrosyringe. At day 3, the area surrounding each injection site wasexcised, prepared for histochemical analysis, stained forβ-galactosidase to determine the location and viability of the cellscarrying the LacZ marker, examined microscopically, and photographed.The results of these experiments demonstrate that MDC compositions canbe used as myocardial soft tissue augmentation materials (FIGS. 7A and7B) for the treatment of injury or weakness secondary to heart failureor myocardial infarction.

Example 8 MDC Infection into Liver, Spleen, and Spinal Cord Tissues

SD rats were prepared for surgery as described above. A midline abdomenincision was made to expose the liver and spleen. Both sites wereinjected with 10 μl of MDC suspension in HBSS (1-1.5×10⁶ cells) using aHamilton microsyringe. At the same time, a midline back incision and apartial laminectomy was made to expose the spinal cord. Spinal cordtissues at level T10 were then injected with the MDC suspension in HBSSas done for the liver and spleen tissues. At day 4, the area surroundingeach injection site was excised, prepared histochemical analysis,stained for β-galactosidase to determine the location and viability ofthe cells carrying the LacZ marker, examined microscopically, andphotographed. These experiments show that MDC compositions can be usedas liver, spleen, and spinal cord soft tissue augmentation materials(FIGS. 8A-8B, 9A-9B, and 10A-10B) to treat various liver, spleen, andspinal cord injuries, diseases, or dysfunctions.

Example 9 MDC Treatment of Bone Defects

Isolation of muscle derived cells: MDC were obtained from mdx mice asdescribed in Example 1.

Clonal isolation of PP6 muscle-derived progenitor cells: To isolateclones from the PP6 cell population, PP6 cells were transfected with aplasmid containing the LacZ, mini-dystrophin, and neomycin resistancegenes. Briefly, a SmaI/SalI fragment containing the neomycin resistancegene from pPGK-NEO was inserted into the SmaI/SalI site in pIEPlacZplasmid containing the LacZ gene, creating the pNEOlacZ plasmid. TheXhoI/SalI fragment from DysM3 which contains the short version of thedystrophin gene (K. Yuasa et al., 1998, FEBS Lett. 425:329-336; giftfrom Dr. Takeda, Japan) was inserted into SalI site in the pNEOlacZ togenerate a plasmid which contains the mini-dystrophin, LacZ, andneomycin resistance genes. The plasmid was linearized by SalI digestionprior to transfection.

PP6 cells were transfected with 10 μg of the linear plasmid containingmini-dystrophin, LacZ, and neomycin resistance gene using theLipofectamine Reagent (Gibco BRL) according to the manufacturer'sinstructions. At 72 hours after transfection, cells were selected with3000 μg/ml of G418 (Gibco BRL) for 10 days until discrete coloniesappeared. Colonies were then isolated and expanded to obtain a largequantity of the transfected cells, and then tested for expression ofLacZ. One of these PP6-derived clones, mc13, was used for further study.

Immunohistochemistry: PP6, mc13, and mouse fibroblast cells were platedin a 6-well culture dish and fixed with cold methanol for 1 minute.Cells were then washed with phosphate buffered saline (PBS), and blockedwith 5% horse serum at room temperature for 1 hour. The primaryantibodies were diluted in PBS as follows: anti-desmin (1:100, Sigma),biotinylated anti-mouse CD34 (1:200, Pharmingen), rabbit anti-mouseBcl-2 (1:500, Pharmingen), rabbit anti-mouse M-cadherin (1:50, gift fromDr. A. Wernig), mouse anti-mouse MyoD (1:100, Pharmingen), mouseanti-rat myogenin (1:100, Pharmingen), rabbit anti-mouse Flk-1 (1:50,Research Diagnostics), and biotinylated Sca-1 (1:100, Pharmingen). Cellswere incubated with the primary antibodies at room temperatureovernight. Cells were then washed and incubated with the appropriatebiotinylated secondary antibodies for 1 hour at room temperature.Subsequently, the cells were rinsed with PBS then incubated at roomtemperature with 1/300 streptavidin conjugated with Cy3 fluorochrome for1 hour. Cells were then analyzed by fluorescence microscopy. For eachmarker, the percentage of stained cells was calculated for 10 randomlychosen fields of cells.

Cryosections of muscle samples from a four week old normal mouse (C-57BL/6J, Jackson Laboratories) were fixed with cold acetone for 2 minutesand pre-incubated in 5% horse serum diluted in PBS for 1 hour. For CD34,Bcl-2, and collagen type IV, the following primary antibodies were used:biotin anti-mouse CD34 (1:200 in PBS, Pharmingen), rabbit anti-mouseBcl-2 (1:1000, Pharmingen), and rabbit anti-mouse collagen type IV(1:100 in PBS, Chemicon). For dystrophin staining, sheep-anti-human DY10antibody (1:250 dilution in PBS) was used as the primary antibody, andthe signal was amplified using anti-sheep-biotin (1:250 dilution inPBS), and streptavidin-FITC (1:250 dilution in PBS).

Stimulation with rhBMP-2, osteocalcin staining, and alkaline phosphataseassay: Cells were plated in triplicate at a density of 1-2×10⁴ cells perwell in 12 well collagen-coated flasks. The cells were stimulated by theaddition of 200 ng/ml recombinant human BMP-2 (rhBMP-2) to the growthmedium. The growth medium was changed on days 1, 3, and 5 following theinitial plating. A control group of cells was grown in parallel withoutadded rhBMP-2. After 6 days with or without rhBMP-2 stimulation, cellswere counted using a microcytometer and analyzed for osteocalcin andalkaline phosphatase expression. For osteocalcin staining, cells wereincubated with goat anti-mouse osteocalcin antibodies (1:100 in PBS,Chemicon), followed by incubation with anti-goat antibodies conjugatedwith the Cy3 fluorochrome. To measure alkaline phosphatase activity,cell lysates were prepared and analyzed using a commercially availablekit that utilizes color change in the reagent due to the hydrolysis ofinorganic phosphate from p-nitrophenyl phosphate (Sigma). The resultingcolor change was measured on a spectrophotometer, and the data wereexpressed as international units ALP activity per liter normalized to10⁶ cells. Statistical significance was analyzed using student's t-test(p<0.05).

In vivo differentiation of mc13 cells in myogenic and osteogeniclineages—Myogenic: The mc13 cells (5×10⁵ cells) were injectedintramuscularly in the hind limb muscle of mdx mice. The animals weresacrificed at 15 days post-injection, and the injected muscle tissue wasfrozen, cryostat sectioned, and assayed for dystrophin (see above) andLacZ expression. To test for LacZ expression, the muscle sections werefixed with 1% glutaraldehyde and then were incubated with X-galsubstrate (0.4 mg/ml 5-bromochloro-3 indolyl-β-D-galactoside(Boehringer-Mannheim), 1 mM MgCl₂, 5 mM K₄Fe(CN)₆, and 5 mM K₃Fe(CN)₆ inphosphate buffered saline) for 1-3 hours. Sections were counter-stainedwith eosin prior to analysis. In parallel experiments, mc13 cells (5×10⁵cells) were injected intravenously in the tail vein of mdx mice. Theanimals were sacrificed at 7 days post-injection and hind limbs wereisolated and assayed for the presence of dystrophin and β-galactosidaseas described.

Osteogenic: To construct the adenovirus BMP-2 plasmid (adBMP-2), therhBMP-2 coding sequence was excised from the BMP-2-125 plasmid (GeneticsInstitute, Cambridge, Mass.) and subcloned into a replication defective(E1 and E3 gene deleted) adenoviral vector containing the HuCMVpromoter. Briefly, the BMP-2-125 plasmid was digested with SalI,resulting in a 1237 base pair fragment containing the rhBMP-2 cDNA. TherhBMP-2 cDNA was then inserted into the SalI site of the pAd.loxplasmid, which placed the gene under the control of the HuCMV promoter.Recombinant adenovirus was obtained by co-transfection of pAd.lox withpsi-5 viral DNA into CRE-8 cells. The resulting adBMP-2 plasmid wasstored at −80° C. until further use.

Mc13 cells were trypsinized and counted using a microcytometer prior toinfection. Cells were washed several times using HBSS (GibcoBRL).Adenovirus particles equivalent to 50 multiplicity of infection unitswere premixed into HBSS then layered onto the cells. Cells wereincubation at 37° C. for 4 hours, and then incubated with an equalvolume of growth medium. Injections of 0.5-1.0×10⁶ cells were performedusing a 30-gauge needle on a gas-tight syringe into exposed tricepssurae of SCID mice (Jackson Laboratories). At 14-15 days, the animalswere anesthetized with methoxyflurane and sacrificed by cervicaldislocation. The hind limbs were analyzed by radiography. Subsequently,the triceps surae were isolated and flash frozen in 2-methylbutanebuffered in phosphate buffered saline, and pre-cooled in liquidnitrogen. The frozen samples were cut into 5-10 μm sections using acryostat (Microm, HM 505 E, Fisher Scientific) and stored at −20° C. forfurther analysis.

RT-PCR analysis: Total RNA was isolated using TRIzol reagent (LifeTechnologies). Reverse transcription was carried out using SuperScript™Preamplification System for First Strand cDNA Synthesis (LifeTechnologies) according to the instructions of the manufacturer.Briefly, 100 ng random hexamers were annealed to 1 μg total RNA at 70°C. for 10 minutes, and then chilled on ice. Reverse transcription wascarried out with 2 μl 10×PCR buffer, 2 μl 25 mM MgCl₂, 1 μl 10 mM dNTPmix, 2 μl 0.1 M DTT, and 200 U superscript II reverse transcriptase. Thereaction mixture was incubated for 50 minutes at 42° C.

Polymerase chain reaction (PCR) amplification of the targets wasperformed in 50 μl reaction mixture containing 2 μl of reversetranscriptase reaction product, 100 μl (5 U) Taq DNA polymerase (LifeTechnologies), and 1.5 mM MgCl₂. The CD34 PCR primers were designedusing Oligo software and had the following sequences: CD34 UP: TAA CTTGAC TTC TGC TAC CA (SEQ ID NO:1); and CD34 DOWN: GTG GTC TTA CTG CTG TCCTG (SEQ ID NO:2). The other primers were designed according to previousstudies (J. Rohwedel et al., 1995, Exp. Cell Res. 220:92-100; D. D.Cornelison et al., 1997, Dev. Biol. 191:270-283), and had the followingsequences: C-MET UP: GM TGT CGT CCT ACA CGG CC (SEQ ID NO:3); C-METDOWN: CAC TAC ACA GTC AGG ACA CTG C (SEQ ID NO:4); MNF UP: TAC TTC ATCAAA GTC CCT CGG TC (SEQ ID NO:5); MNF DOWN: GTA CTC TGG MC AGA GGC TAACTT (SEQ ID NO:6); BCL-2 UP: AGC CCT GTG CCA CCA TGT GTC (SEQ ID NO:7);BCL-2 DOWN: GGC AGG TTT GTC GAC CTC ACT (SEQ ID NO:8); MYOGENIN UP: CMCCA GGA GGA GCG CGA TCT CCG (SEQ ID NO:9); MYOGENIN DOWN: AGG CGC TGTGGG AGT TGC ATT CAC T (SEQ ID NO:10); MYOD UP: GCT CTG ATG GCA TGA TGGATT ACA GCG (SEQ ID NO:11); and MYOD DOWN: ATG CTG GAC AGG CAG TCG AGG C(SEQ ID NO:12).

The following PCR parameters were used: 1) 94° C. for 45 seconds; 2) 50°C. for 60 seconds (CD34) or 60° C. for 60 seconds (for myogenin andc-met); and 3) 72° C. for 90 seconds for 40 cycles. PCR products werechecked by agarose-TBE-ethidium bromide gels. The sizes of the expectedPCR products are: 147 bp for CD34; 86 bp for myogenin; and 370 bp forc-met. To exclude the possibility of genomic DNA contamination, twocontrol reactions were completed: 1) parallel reverse transcription inthe absence of reverse transcriptase, and 2) amplification of β-actinusing an intron-spanning primer set (Clonetech).

Skull defect assay: Three 6-8 week old female SCID mice (JacksonLaboratories) were used in control and experimental groups. The animalswere anesthetized with methoxyflurane and placed prone on the operatingtable. Using a number 10 blade, the scalp was dissected to expose theskull, and the periosteum was stripped. An approximately 5 mmfull-thickness circular skull defect was created using a dental burr,with minimal penetration of the dura. A collagen sponge matrix(Helistat™, Colla-Tec, Inc.) was seeded with 0.5-1.0×10⁶ MDC either withor without adBMP-2 transduction, and placed into the skull defect. Thescalp was closed using a 4-0 nylon suture, and the animals were allowedfood and activity. After 14 days, the animals were sacrificed, and theskull specimens were observed and then analyzed microscopically. For vonKossa staining, skull specimens were fixed in 4% formaldehyde and thensoaked in 0.1 M AgNo₃ solution for 15 minutes. The specimens wereexposed to light for at least 15 minutes, washed with PBS, and thenstained with hematoxylin and eosin for viewing.

Fluorescence in situ hybridization using Y-probes: The cryosections werefixed for 10 minutes in 3:1 methanol/glacial acetic acid (v:v) and airdried. The sections were then denatured in 70% formamide in 2×SSC (0.3 MNaCl, 0.03 M NaCitrate) pH 7.0 at 70° C. for 2 minutes. Subsequently,the slides were dehydrated with a series of ethanol washes (70%, 80%,and 95%) for 2 minutes at each concentration. The Y-chromosome specificprobe (Y. Fan et al., 1996, Muscle Nerve 19:853-860) was biotinylatedusing a BioNick kit (Gibco BRL) according to the manufacturer'sinstructions. The biotinylated probe was then purified using a G-50Quick Spin Column (Boehringer-Mannheim), and the purified probe waslyophilized along with 5 ng/ml of sonicated herring sperm DNA. Prior tohybridization, the probe was re-suspended in a solution containing 50%formamide, 1×SSC, and 10% dextran sulfate. After denaturation at 75° C.for 10 minutes, the probe was placed on the denatured sections andallowed to hybridize overnight at 37° C. After hybridization, thesections were rinsed with 2×SSC solution pH 7.0 at 72° C. for 5 minutes.The sections were then rinsed in BMS solution (0.1 M NaHCO₃, 0.5 M NaCl,0.5% NP-40, pH 8.0). The hybridized probe was detected with fluoresceinlabeled avidin (ONCOR, Inc). The nuclei were counter-stained with 10ng/ml ethidium bromide in Vectashield mounting medium (Vector, Inc).

Marker analysis of mc13 cells: The biochemical markers expressed bymc13, PP6, and fibroblast cells were analyzed using RT-PCR andimmunohistochemistry. Table 3 (below) shows that mc13 cells expressedFlk-1, a mouse homologue of the human KDR gene, which was recentlyidentified as a marker of hematopoietic cells with stem cell-likecharacteristics (B. L. Ziegler et al., supra), but did not express CD34or CD45. However, other clonal isolates derived from the PP6 MDC of thepresent invention expressed CD34, as well as other PP6 cell markers. Itwill be appreciated by those skilled in the art that the proceduresdescribed herein can be used to clone out the PP6 muscle-derivedprogenitor cell population and obtain clonal isolates that express cellmarkers characteristic of the muscle-derived progenitor cells. Suchclonal isolates can be used in accordance with the methods of theinvention. For example, the clonal isolates express progenitor cellmarkers, including desmin, CD34, and Bcl-2. Preferably, the clonalisolates also express the Sca-1 and Flk-1 cell markers, but do notexpress the CD45 or c-Kit cell markers.

TABLE 3 Table 3: Cell markers expressed by mdx PP6, mdx mc13, andfibroblast cells. PP6 cells MC13 cells Fibroblasts imm RT-PCR imm RT-PCRimm RT-PCR desmin + na + na − na CD34 + + − − − − Bcl-2 + na +/− na − naFlk-1 + na + na − na Sca-1 + na + na − na M-cadherin −/+ na + na − naMyogenin +/− + +/− + − − c-met na + na + na − MNF na + na + na − MyoD−/+ + na + na − c-Kit − na − na na na CD45 − na − na na na Cells wereisolated as described above and examined by immunohistochemicalanalysis. “−” indicates that 0% of the cells showed expression; “+”indicates that >98% of the cells showed expression; “+/−” indicates that40-80% of the cells showed expression; “−/+” indicates that 5-30% of thecells showed expression; “na” indicates that the data is not available.

In vivo localization of CD34⁺ and Bcl-2⁺ cells: To identify the locationof CD34+ and Bcl-2+ cells in vivo, muscle tissue sections from thetriceps surae of normal mice were stained using anti-CD34 and anti-Bcl-2antibodies. The CD34 positive cells constituted a small population ofmuscle derived cells (FIG. 12A) that were also positive for desmin (FIG.12B). Co-staining the CD34+, desmin+ cells with anti-collagen type IVantibody localized them within the basal lamina (FIGS. 128 and 12D). Asindicated by the arrowheads in FIGS. 12A-D, small blood vessels werealso positive for CD34 and collagen type IV, but did not co-localizewith the nuclear staining. The expression of CD34 by vascularendothelial cells has been shown in previous studies (L. Fina et al.,supra). The Bcl-2+, desmin+ cells were similarly identified (FIGS.12E-12H) and localized within the basal lamina (FIGS. 12F and 12H). Thesections were also stained for M-cadherin to identify the location ofsatellite cells (FIG. 12I). The satellite cells were identified atsimilar locations as CD34+, desmin+, or Bcl-2+, desmin+ cells (arrow,FIG. 12I). However, multiple attempts to co-localize CD34 or Bcl-2 withM-cadherin were unsuccessful, suggesting that M-cadherin expressingcells do not co-express either Bcl-2 or CD34. This is consistent withPP6 cells expressing high levels of CD34 and Bcl-2, but expressingminimal levels of M-cadherin, as disclosed herein.

In vitro differentiation of clonal muscle progenitor cells intoosteogenic lineage: Mc13 cells were assessed for osteogenicdifferentiation potential by stimulation with rhBMP-2. Celli were platedon 6-well culture dishes and grown to confluency in the presence orabsence of 200 ng/ml rhBMP-2. Within 3-4 days, mc13 cells exposed torhBMP-2 showed dramatic morphogenic changes compared to cells withoutrhBMP-2. In the absence of rhBMP-2, mc13 cells began to fuse intomultinucleated myotubes (FIG. 13A). When exposed to 200 ng/ml rhBMP-2,however, cells remained mononucleated and did not fuse (FIG. 13B). Whencell density reached >90% confluency, the untreated culture fused toform multiple myotubes (FIG. 13C), while the treated cells becamecircular and hypertrophic (FIG. 13D). Using immunohistochemistry, thesehypertrophic cells were analyzed for the expression of osteocalcin.Osteocalcin is a matrix protein that is deposited on bone, specificallyexpressed by osteoblasts. In contrast to the untreated group, therhBMP-2 treated hypertrophic cells showed significant expression ofosteocalcin (FIG. 13E), thus suggesting that mc13 cells are capable ofdifferentiating into osteoblasts upon exposure to rhBMP-2.

Mc13 cells were then analyzed for expression of desmin following rhBMP-2stimulation. Newly isolated mc13 cells showed uniform desmin staining(FIGS. 14A and 14B). Within 6 days of exposure to rhBMP-2, only 30-40%of mc13 cells showed desmin staining. In the absence of rhBMP-2stimulation, approximately 90-100% of mc13 cells showed desmin staining(FIG. 14C). This result suggests that stimulation of mc13 cells withrhBMP-2 results in the loss of myogenic potential for these cells.

In addition, mc13 cells were analyzed for the expression of alkalinephosphatase following rhBMP-2 stimulation. Alkaline phosphatase has beenused as a biochemical marker for osteoblastic differentiation (T.Katagiri et al., 1994, J. Cell Biol. 127:1755-1766). As shown in FIG.14D, alkaline phosphatase expression of mc13 cells was increased morethan 600 fold in response to rhBMP-2. PP1-4 cells, used as a control,did not show increased alkaline phosphatase activity in response torhBMP-2 (FIG. 14D). Taken together, these data demonstrate that cells ofa PP6 clonal isolate, e.g., mc13 cells, can lose their myogenic markersand differentiate through the osteogenic lineage in response to rhBMP-2exposure in vitro.

In vivo differentiation of mc13 cells into myogenic and osteogeniclineages: To determine whether mc13 cells were capable ofdifferentiating through the myogenic lineage in vivo, the cells wereinjected into the hind limb muscle tissue of mdx mice. The animals weresacrificed 15 days following injection, and their hind limbs wereharvested for histological and immunohistochemical analysis. Severalmyofibers showed LacZ and dystrophin staining in the region surroundingthe injection site (FIGS. 15A and 15B), indicating that mc13 cells candifferentiate through the myogenic lineage in vivo and enhance muscleregeneration and restore dystrophin in the dystrophic muscle.

In a parallel experiment, mc13 cells were injected intravenously intothe tail vein of mdx mice. The animals were sacrificed at 7 dayspost-injection, and the hind limb muscles were harvested forhistological and immunohistochemical analysis. Several hind limb musclecells showed LacZ and dystrophin staining (FIGS. 15C-15D; see also “*”),suggesting that mc13 cells can be delivered systemically to the targettissue for rescue of dystrophin expression.

To test the pluripotent characteristics of mc13 cells in vivo, the cellswere transduced with an adenoviral vector encoding rhBMP-2 (adBMP-2).The mc13 cells with adBMP-2 were then injected into hind limbs of SCIDmice. The animals were sacrificed at 14 days post-injection, and thehind limbs were removed for histochemical and immunochemical analysis.Enzyme-linked immunosorbent assay (ELISA) analysis of mc13 cellstransduced with adBMP-2 showed that infected cells were capable ofproducing rhBMP-2. Radiographic analysis of hind limbs of injected SCIDmice revealed robust ectopic bone formation within 14 days of injection(FIG. 15E). Histological analysis using LacZ staining of the ectopicbone shows that LacZ positive mc13 cells were uniformly located withinthe mineralized matrix or lacunae, a typical location where osteoblastsand osteocytes are found (FIG. 15F).

To further confirm the role of mc13 in formation of the ectopic bone,the muscle sections were also stained for presence of dystrophin. Asshown in FIG. 15G, the ectopic bone contained cells highly positive fordystrophin, suggesting that mc13 cells are intimately participating inbone formation. As a control, similar experiments were carried out withfibroblasts. Fibroblasts were found to support robust ectopic boneformation, but the injected cells were uniformly found outside of thebone, and none could be located within the mineralized matrix. Thissuggests that the fibroblasts are capable of delivering rhBMP-2 to formectopic bone, but are unable to differentiate into osteoblasts. In thiscase, the cells participating in mineralization of the ectopic bone aremost likely derived from the host tissue. Thus, these resultsdemonstrate that mc13 cells can differentiate into osteoblasts, both invivo and in vitro,

Enhancement of bone healing by genetically engineered muscle-derivedcells: Skull defects (approximately 5 mm) were created in skeletallymature (6-8 weeks old) female SCID mice using a dental burr as describedabove. Previous experiments have demonstrated that 5 mm skull defectsare “non-healing” (P. H. Krebsbach et al., 1998, Transplantation66:1272-1278). The skull defect was filled with a collagen sponge matrixseeded with mc13 cells transduced or not transduced with adBMP-2. Thesemice were sacrificed at 14 days, and the healing of the skull defect wasanalyzed. As shown in FIG. 16A, the control group treated with mc13cells without rhBMP-2 showed no evidence of healing of the defect. Incontrast, the experimental group treated with mc13 cells transduced toexpress rhBMP-2 showed almost a full closure of the skull defect at 2weeks (FIG. 16B). The von Kossa staining, which highlights mineralizedbone, showed robust bone formation in the group treated with mc13 cellstransduced to express rhBMP-2 (FIG. 16D), but minimal bone formation wasobserved in the control group (FIG. 16C).

The area of new bone in the experimental group was analyzed byfluorescence in situ hybridization (FISH) with a Y-chromosome specificprobe to identify transplanted cells. As shown in FIG. 16E, Y-chromosomepositive cells were identified within the newly formed bone, indicatingactive participation of transplanted cells in bone formation under theinfluence of rhBMP-2. The Y-chromosome negative cells were alsoidentified within the newly formed skull, thus indicating activeparticipation of host-derived cells as well. These results demonstratethat mc13 cells can mediate healing of a “non-healing” bone defect uponstimulation with rhBMP-2, and indicate that the MDC of the presentinvention can be used in the treatment of bone defects, injuries, ortraumas.

All patent applications, patents, texts, and literature references citedin this specification are hereby incorporated herein by reference intheir entirety to more fully describe the state of the art to which thepresent invention pertains.

As various changes can be made in the above methods and compositionswithout departing from the scope and spirit of the invention asdescribed, it is intended that all subject matter contained in the abovedescription, shown in the accompanying drawings, or defined in theappended claims be interpreted as illustrative, and not in a limitingsense.

What is claimed is:
 1. A method of augmenting or bulking the analsphincter soft-tissue in a recipient, comprising: introducing anisolated population of cultured cells enriched in viable,desmin-expressing, skeletal muscle-derived myoblasts into the analsphincter soft-tissue of the recipient in an amount sufficient for themyoblasts to augment or bulk the anal sphincter soft-tissue; wherein theskeletal muscle-derived myoblasts are introduced in a compositioncomprising a physiologically acceptable medium, wherein the skeletalmuscle-derived myoblasts are isolated using a method comprising: (a)plating a suspension of skeletal muscle cells from skeletal muscletissue in a first container containing cell culture media, whereinfibroblast cells of the skeletal muscle cell suspension adhere to thewall of the first container; (b) re-plating non-adherent cells from step(a) in a second container containing media; (c) re-plating thenon-adherent cells from step (b) serially in 4 containers containingmedia, after 30-40% of cells have adhered to each container; and (d)isolating the skeletal muscle-derived myoblasts from the wall of thesixth container following the re-plating in step (c), wherein theisolated cells are CD34⁺, Bcl-2⁺, KDR⁺, and Sca-1⁺.
 2. The methodaccording to claim 1, wherein the composition is introduced into therecipient by an administration route selected from injection orintravenous delivery.
 3. The method according to claim 1, wherein thecomposition further comprises a carrier, excipient, or diluent.
 4. Themethod according to claim 3, wherein the carrier comprises an absorbentor adherent material.
 5. The method according to claim 3, wherein thecarrier is a collagen sponge material.
 6. The method according to claim1, wherein the skeletal muscle-derived myoblasts arehistocompatibly-matched with the recipient.
 7. A method of treatingfecal incontinence in a subject in need thereof, comprising introducingan isolated population of cultured cells enriched in viable,desmin-expressing, skeletal muscle-derived myoblasts into the analsphincter soft-tissue of the recipient in an amount sufficient for themyoblasts to augment or bulk the anal sphincter soft-tissue; wherein theskeletal muscle-derived myoblasts are introduced in a compositioncomprising a physiologically acceptable medium, wherein the skeletalmuscle-derived myoblasts are isolated using a method comprising: (a)plating a suspension of skeletal muscle cells from skeletal muscletissue in a first container containing cell culture media, whereinfibroblast cells of the skeletal muscle cell suspension adhere to thewall of the first container; (b) re-plating non-adherent cells from step(a) in a second container containing media; (c) re-plating thenon-adherent cells from step (b) serially in 4 containers containingmedia, after 30-40% of cells have adhered to each container; and (d)isolating the skeletal muscle-derived myoblasts from the wall of thesixth container following the re-plating in step (c), wherein theisolated cells are CD34⁺, Bcl-2⁺, KDR⁺, and Sca-1⁺.
 8. The methodaccording to claim 7, wherein the composition is introduced into therecipient by an administration route selected from injection orintravenous delivery.
 9. The method according to claim 8, wherein thecomposition further comprises a carrier, excipient, or diluent.
 10. Themethod according to claim 9, wherein the carrier comprises an absorbentor adherent material.
 11. The method according to claim 10, wherein thecarrier is a collagen sponge material.
 12. The method according to claim8, wherein the skeletal muscle-derived myoblasts arehistocompatibly-matched with the recipient.